A Recombinant Fab Antibody Against Kwakhurin as a Tool for Sensitive Indirect Competitive ELISA

Chanpokapaiboon, Kaskamol; Khoonrit, Pichayatri; Yusakul, Gorawit; Juengwatanatrakul, Thaweesak; Putalun, Waraporn; Tanaka, Hiroyuki; Sakamoto, Seiichi; Morimoto, Satoshi

doi:10.2174/1389201020666181226105223

Cited by 5 publications

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“…The recombinant antigen-binding fragment (Fab) antibody-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed, which showed selectivity and sensitivity for Kwa [limit of detection (LOD) 8.16 ng/mL]. 11 The monoclonal antibody (IgG)-based icELISA was developed for the standardization of P. candollei materials, in which the sensitivity was higher than in the previous report (LOD 1.13 ng/mL). Recently, a new format of icELISA was invented using Kwa magnetic particle conjugates, which also provided high sensitivity (LOD 1.90 ng/mL) and a faster time of analysis than the conventional icELISA.…”

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confidence: 99%

Immunochromatographic assay for the detection of kwakhurin and its application for the identification of Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham

Phaisan

Yusakul

Nuntawong

et al. 2020

Phytochemical Analysis

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Introduction: The plant Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham (PM), known by its common Thai name as white Kwao Krua, is sometimes misidentified because it presents similar botanical characteristics to those of Butea superba (red Kwao Krua). The phytochemicals in PM are phytoestrogens in the class of isoflavonoids, but Butea superba contains flavonoids that exhibit androgenic and antiestrogen effects.Objectives: This research aims to develop a simple analytical method for identification and to differentiate PM from red Kwao Krua and other Pueraria species.Methods: A gold nanoparticle-based immunochromatographic assay (ICA) was developed for the detection of kwakhurin (Kwa), a unique compound found in PM. The parameters, including sensitivity, accuracy, precision, and specificity, were validated. All samples were analyzed using ICA and high-performance liquid chromatography with UV detector (HPLC-UV). The results of the two methods were compared for consistency checking. Results:The cutoff limit of Kwa detection was 160 ng/mL, which was lower than in the HPLC-UV method. The repeatability and reproducibility of the ICA preparation and assembly showed high precision. The cross-reactivity to related isoflavonoids was less than 0.32%, which implied high specificity of the ICA for Kwa. Moreover, false-positive and false-negative results from other plant extracts were not observed. Conclusion:The developed ICA is applicable for distinguishing PM from red Kwao Krua and other Pueraria species. This simple analytical method can be applied for the identification of raw PM materials in the industrial and agricultural sectors.

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Immunochromatographic assay for the detection of kwakhurin and its application for the identification of Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham

Phaisan

Yusakul

Nuntawong

et al. 2020

Phytochemical Analysis

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“…In our previous study, we focused on Kwa as a marker compound for standardization as it is a phytoestrogen [ 13 ] produced solely by P. candollei . We also developed various immunoassays for the determination of Kwa in P. candollei -derived products [ 15 , 16 , 17 , 18 ] as immunoassays enable sensitive and selective analyses of small molecules [ 19 , 20 ]. However, an animal-based approach is necessary for the production of antibodies used in immunoassays.…”

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confidence: 99%

Bioimprinting as a Receptor for Detection of Kwakhurin

et al. 2022

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Bioimprinting was performed against ovalbumin (OVA) to confer its binding cavities for kwakhurin (Kwa), an isoflavonoid, produced solely by Pueraria candollei var. mirifica (P. candollei). The characterization of bioimprinted-OVA (biOVA), evaluated by an enzyme-linked immunosorbent assay (ELISA), revealed that it functioned as a specific receptor for Kwa. Using biOVA, two systems, i.e., an indirect competitive ELISA (icELISA) and the even simpler and more rapid competitive enzyme-linked bioimprinted-protein assay (cELBIA), were developed as novel techniques for the quantitative analysis of Kwa in P. candollei and its related products. The two analysis methods were found to have limits of detection (LOD) of 4.0 and 2.5 µg/mL, respectively. The high reliability of the developed icELISA and cELBIA using biOVA was also demonstrated by various validation analyses. Subsequently, bioimprinting was performed using eight other proteins to investigate them as candidate scaffolds for the generation of binding cavities for Kwa. Interestingly, two bioimprinted-IgG monoclonal antibodies (biMAbs) recognized Kwa, but their original binding affinity to hapten was lost. That is, the MAbs obtained a new binding ability to Kwa in exchange for their original binding affinity, raising the possibility that biMAb could be alternatively used as a probe for the quantitative analysis of Kwa as well as biOVA. This is the first report of small molecules recognition by MAbs used as proteins for bioimprinting.

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“…In addition, the assembly between V H ‐C H 1 and LC of anti‐paclitaxel Fab (IgG2a) was also not efficient in both Escherichia coli and Bombyx mori nuclear polyhedrosis virus (BmNPV) bacmid expression systems . Fragments of HC (IgG1) and LC were well folded and associated with the structure of a Fab using in vitro refolding, transgenic potato plants, Chinese hamster ovary cells, B. mori larvae, and P. pastoris …”

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confidence: 99%

Modification of the first constant domain of heavy chain enabled effective folding of functional anti‐forskolin antigen‐binding fragment for sensitive quantitative analysis

Yusakul

Sakamoto

Tanaka

et al. 2019

Biotechnology Progress

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The assembly between heavy and light chains is a critical step of immunoglobulin (Ig) and fragment antigen‐binding (Fab) antibody expression and of their binding activity. The genes encoding Fab were obtained from hybridoma cells secreting monoclonal antibody (MAb, IgG2b) against adenylate cyclase activator forskolin (FOR). The subclass of the first constant domain of heavy chain (CH1) of IgG2b was modified to IgG1 via overlap extension polymerase chain reaction and expressed via Escherichia coli bacterial system. Since both Fabs (IgG2b and IgG1) were expressed as inclusion bodies, functional analysis was performed after in vitro refolding via stepwise dialysis. The result indicated that the folding efficiency between VH‐CH1 and VL‐CL was improved by the CH1 modification from IgG2b to IgG1 subclass, although their specificity for FOR was not altered. Effective folding of IgG1 was also observed when they were expressed in the hemolymph of silkworm larvae using the Bombyx mori nuclear polyhedrosis virus bacmid system. An indirect competitive enzyme‐linked immunosorbent assay (icELISA) was then developed for the determination of FOR using effectively prepared Fab IgG1. The sensitivity of FOR determination was in the range of 3.91–62.5 ng/mL with less than 9% relative standard deviation, implying the sensitive and reliable analysis of developed icELISA. In addition, high accuracy of the icELISA was supported by the results of spiked‐and‐recovery tests, ranging from 100.2 to 102.3%. Therefore, Fab could be utilized reliably for icELISA instead of the more expensive MAb. Collectively, this approach improved productivity of Fab and reduced the cost of antibody production.

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A Recombinant Fab Antibody Against Kwakhurin as a Tool for Sensitive Indirect Competitive ELISA

Cited by 5 publications

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Immunochromatographic assay for the detection of kwakhurin and its application for the identification of Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham

Immunochromatographic assay for the detection of kwakhurin and its application for the identification of Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham

Bioimprinting as a Receptor for Detection of Kwakhurin

Modification of the first constant domain of heavy chain enabled effective folding of functional anti‐forskolin antigen‐binding fragment for sensitive quantitative analysis

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